Sodium/Calcium exchangers (NCX) play a key role in muscle contraction of cardiac myocytes in the vertebrate heart. Human NCX1 is expressed at high levels in the plasma membrane of the transverse (t)- tubules of cardiac myocytes and couples the electrogenic efflux of one Ca2+ to the import of 3 Na+ ions during the relaxation phase of muscle contraction. The importance of NCX1 in regulating intracellular Ca2+ levels in the heart is underscored by the embryonic lethality of NCX1 knockout mice as well as the numerous pathological conditions associated with aberrant Ca2+ transport, including cardiac arrythmias and heart failure. Although there is a large body of literature on NCX1 and its homologs, including electrophysiological, immunochemical, and pharmacological studies, there is no detailed structural information on these proteins to date. As a result, high resolution structural data is essential to understand the molecular mechanism of NCX proteins. As there are NCX1 homologs present in bacteria and archaea, these proteins may provide an avenue to the high-resolution structural information currently unavailable in present studies. The aims of the research proposed in this application are: 1.) To express and purify adequate amounts of prokaryotic NCX1 homologs for structural studies and assess their monodispersity and biochemical activity in proteoliposomes;and 2.) To obtain high resolution structural information of a prokaryotic NCX protein by X-ray crystallography. These studies could provide invaluable details unavailable by current methods, including the stereochemistry of the ion binding sites and the nature of the conformational changes associated with transport. Furthermore, knowledge of the functional architecture of an NCX protein could aid homology modeling and hence structure-based pharmaceutical design of potential inhibitors of the human NCX family members. PUBLIC HEALTH RELEVANCE. The rise and fall of Ca2+ levels are coupled to the rhythmic muscle contraction of the beating heart, and these levels are directly controlled by the activity of the Na+/Ca2+ exchanger protein (NCX). As abnormal Ca2+ levels have been linked to various cardiac pathologies, including arrythimias and heart failure, studies of the mechanism of NCX could shed light on the detailed causes of these diseases.